Transmissible spongiform encephalopathies (“TSEs”) are fatal neurodegenerative diseases presenting as inherited, sporadic, or infectious forms, with the accumulation of an misfolded, protease resistant form of prion protein (PrPSc) in the central nervous system as their pathological underpinning. Examples of disorders in this group include kuru, Creutzfeldt-Jakob disease (CJD), and variant CJD in humans; scrapie in sheep and goats; bovine spongiform encephalopathy in cattle; transmissible mink encephalopathy in mink; and chronic wasting disease in cervids.
The pathobiology, specifically the transmission of and mechanism for infection of transmissible spongiform encephalopathies is not well understood. One hypothesis presumes that a protein confirmation change converts the normal cellular form of the prion protein (PrPC) into disease-associated PrPSc. However, it remains uncertain whether PrPSc is a TSE infectious agent and sole reliance on PrPSc may not be a reliable marker for TSE infectivity. (Barron, R., et al., 2007. J. Biol. Chem., 282:35878-35886.) Investigation into the pathobiology of TSEs is further complicated when considering the effects of PrPSc accumulation in natural versus non-natural host species. For example, scrapie-affected sheep (natural host) with demonstrable retinal PrPSc accumulation by immunohistochemistry do not appear to have associated major morphological changes in their retinas when corresponding hematoxylin and eosin stained sections are examined (Greenlee et al., 2006), whereas retinas from scrapie-affected hamsters (non-natural host) exhibit extensive photoreceptor degeneration (Buyukmichi, N., et al., 1980. Proc. Natl. Acad. Sci. USA, Vol. 77, No. 2, 1169-1171; Hogan, R. N., et al., 1981. Lab Invest, 44:34-42.).
There is an increased importance placed on studying TSEs in food-producing livestock including scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, and chronic waste disease (CWD) in mule deer and elk. For instance, it has been hypothesized that the novel variant form of Creutzfeldt-Jakob disease transmitted to humans as a result of exposure to a BSE agent. Given the uncertainty of the transmissible agent, there is a need to develop detection methods of detection to identify and quarantine livestock before they exhibit clinical signs to prevent transmission at an early stage. One of the factors compounding identifying infected subjects is that incubation for TSEs is relatively long before a showing of clinical signs. Clinical signs of TSEs are usually neurological symptoms and for BSEs, signs include hyperesthesia, hindlimb ataxia, pelvic swaying, hypermetria, tremors, falling, recumbency, and behavioral changes such as apprehension, nervousness, and occasionally frenzy. Nonspecific symptoms include loss of condition (wasting), weight loss, and decreased milk production. The incubation period for BSE can be between 30 months to eight years. Given the long incubation period and the importance of livestock, there is a need to develop an antemortem method to detect infected livestock prior to slaughter. Furthermore, early detection of infected livestock would prevent the costly option of slaughtering whole herds of livestock in an effort to quarantine and eliminate infected livestock.
There are postmortem and antemortem screening methods of detecting TSE in livestock. Postmortem detection includes necropsy of subjects showing clinical TSE signs and performing histological and immunohistochemical assays on brain tissue to confirm PrPSc presence. For instance, U.S. Pat. No. 6,261,790 discloses using monoclonal antibodies in an immunological assay as an indication of the presence of PrPSc. Given the unknown agent for TSE transmissibility, it is imperative that early antemortem diagnosis of livestock be conducted to avert further livestock contamination and prevent transmission to humans.
Electroretinograms (“ERGs”) have historically been utilized to detect retinal abnormalities. An electroretinogram is waveform generated by measuring the variation in the electrical potential of the cornea upon photic (light) stimulation. Generally, direct and ground electrodes are applied on or near the subject cornea to record the electrical potential. By altering the visual stimulus and various spatial distributions, ERGs have been utilized to detect deterioration of ganglion cells. (See: U.S. Pat. Nos. 5,539,482, 5,506,633, and 5,382,987 for examples of flashing or pattern alternating stimulation for detecting symptoms related to early glaucoma detection.) Related to TSEs, it is known that PrPSc can be detected in the retina of cattle with BSE. (Bradley, R. et al., 1999. Dev. Biol. Stand., 99:35-40). In scrapie-affected sheep retina, PrPSc accumulation is primarily observed in the inner plexiform layer (IPL), the layer of the retina where synaptic connections occur between retinal bipolar, amacrine, and ganglion cells, and the outer plexiform layer (OPL), where synaptic connections occur between horizontal, bipolar, and photoreceptor cells. In both natural and non-natural host species with TSEs, the retina has been shown to accumulate PrPSc (Bradley, 1999; Foster et al., 1999; Spraker et al., 2002b; Valdez et al., 2003; Head et al., 2003, 2005; Hamir et al., 2004, 2005; Kercher et al., 2004; Hortells et al., 2006; Greenlee et al., 2006). ERGs have been utilized to report a correlation of electroretinographic and histopathologic findings in the eyes of mice infected with scrapie. (Curtis, et al., 1989. Neuropathology and Applied Neurobiology, 15: 75-89.) For humans having clinical signs of Creutzfeldt-Jakob disease, it has been reported that 250 lux flash under both photopic and scotopic conditions led to a decrease in B1-wave and abnormal B/A ratio (J. de Seze, et al., 1998. Neurology, 51:962-967.) No other disclosures of utilizing ERG to detect transmissible spongiform encephalopathies, particularly in livestock, have been reported. As such, there is a need in the field to utilize an electroretinograms system to livestock screen for transmissible spongiform encephalopathies.